1. Field of the Invention
The present invention relates to a new strain of Gliocladium roseum fungus, referred to as ACM941 that is capable of controlling plant diseases. In particular, the present invention relates to the use of the new strain of Gliocladium roseum fungus as a biological control agent (sometimes referred to as bioagent) to inhibit soil-borne and seed-borne fungal pathogens and to enhance plant growth and productivity in both greenhouse and field conditions.
2. Description of Related Art
Soil-borne and seed-borne fungal pathogens of plants are responsible for severe economic losses in the agricultural and horticultural industries worldwide. These pathogens cause plant diseases such as seed decay, root/foot rot, seedling blight and wilt. Such diseases commonly reduce emergence, plant vigor and yield potential. Severe disease infection can kill emerging seedlings of an entire plant population, and result in a total loss of crop yield.
Solutions to the recurring problem of plant pathogens have been explored for decades. As particular crops become more abundant, and the area of land allocated for agriculture expands, there is an inherent need to employ more efficient and effective farming practices. As a result of increasing demand for crop production, farmers must often compromise their cultural practices by planting crops on sub-optimal land, or by increasing the frequency at which crops are planted in a specific location. In doing so, crop nutrients are depleted and specific crop pathogens, especially soil-borne or seed-borne pathogens, become more prevalent. Accordingly, it is increasingly difficult to sustain the health and productivity of a respective crop.
Historically, preferred cultural practices and chemical controls were used in combination to combat destructive pathogens. More recently, the use of integrated systems employing biocontrol agents and chemicals has become more prevalent.1 (note: all superscript reference numerals relate to a list of references appearing at the end of this disclosure). However, despite progressive research in recent years, chemical alternatives remain the most reliable and economic solution to the problems caused by most soil-borne and seed-borne plant pathogens.
The case of field pea is illustrative. There were approximately 900,000 hectares of field pea in western Canada in 1998, estimated at a total farm value of over $400,000 million. In recent years, the field pea crops of western Canada have been most affected by soil-borne or seed-borne diseases, as well as the Ascochyta complexes of root and foliage. Known as PRRC (pea root rot complex) diseases, the soil-borne and seed-borne diseases are most commonly caused by the pathogens Fusarium solani f.sp. pisi, Fusarium oxysporum f.sp. pisi, Mycosphaerella pinodes, Rhizoctonia solani, Sclerotina sclerotiorum, Aphanomyces euteiches, Alternaria alternata and Pythium spp. The incidence of PRRC diseases varies with year and location, while its severity is largely dependent on climate, crop rotation and cultural practices. The yield losses of pea crops alone, as a result of PRRC pathogens including foliar infection by Ascochyta complex, are frequently devastating with a conservative 15% yield reduction translating into an approximate annual loss of $60 million in areas of western Canada. As crops such as pea become increasingly important, efforts must focus on more effective and efficient means of crop farming, both in Canada and around the world.
The most effective solution to the destruction of crop plants by pathogens would most likely be the development of resistant cultivars, which would allow for plant growth and productivity in the presence of fungal pathogens. Unfortunately, success in the development of PRRC resistant cultivars of pea, or other resistant crop cultivars, has not been forthcoming. Alternatively, biological control of crop plant pathogens by microorganisms may be considered a more natural and environmentally friendly alternative to existing chemical treatment methods. Accordingly, it is desirable to isolate a microorganism, which displays antagonistic effects against a target pathogen, and is capable of survival and propagation in a target location.
Efforts to isolate antagonistic microorganisms effective against plant pathogens have been underway in recent years. As a result, several microbial isolates have proven effective as plant pathogen antagonists and some related biocontrol products are currently commercially available, including: Mycostop(trademark) (Streptomyces sp.); GleoGard(trademark) (Trichoderma virens); Kodiak(trademark) (Bacillus sp.); and BioTrek(trademark) (Trichoderma sp.); TRICHODEX(trademark) (Trichoderma harzianum); and BINAB-T(trademark) (T. harzianum plus T. polysporum).
The effectiveness of bioagents against certain pathogens has been characterized according to a variety of modes of action. Cook et al.2 described the modes by which a bioagent can effectively act against target pathogens as including: (i) a parasitic attack against the pathogen, (ii) a competitor for a common food source, (iii) a source of toxic antibiotic substances, or (iv) an induced indirect toxic effect by the release of volatile substances. As such, a bioagent behaves as a natural antagonist to the pathogen.
Although efforts have concentrated on the biological control of PRRC pathogens, obstacles in stability, delivery and versatility have not been resolved. Specifically, Hwang et al.3 in 1992 reported the potential use of Gliocladium virens (Syn. Trichoderma virens) as a biocontrol agent against Rhizoctonia caused root rot in field pea, when employed with a fungicide. In this integrated control system, the presence of the fungicide provided protection against the pathogen when the environmental conditions inhibited the activity of the bioagent. Parke et al.4 in 1991 disclosed findings of the effectiveness of Pseudomonas cepacia and P. fluorescens against Pythium damping-off and Aphanomyces root rot in pea when applied to seed. P. cepacia was disclosed as being the most effective bacterium, increasing emergence by an average of 40% and yield by 48% over captan fungicide alone. The Parke et al. reference further reported on disclosures of the effectiveness of seed treatment with species of Trichoderma5 and Penicilium oxalicum6 against diseases of pea Oyarzun et al.7 further reported findings, in 1993, of the biological control of root rot in pea caused by Fusarium solani, with two nonpathogenic Fusarium oxysporum isolates. Both isolates of F. oxysporum investigated displayed reduced disease severity and prevented the plant weight losses owing to F. solani f. sp. pisi in sterilized soil. In 1996, Xi et al.8 reported on the effectiveness of formulated Rhizobacteria against root rot of field pea. Pseudomonas fluorescens (strain PRA25) peat-based granular formulation increased yield by 17% over untreated, in a trial with light disease infection, and by 120% in another trial with moderate infection. P. cepacia (strain AMMD) and P. fluorescens increased seedling emergence, and decreased disease incidence and severity. However, these agents had variable effect on yield when disease level was light to moderate. In addition, biocoritrol agents resulted in only limited control when disease was severe. As a result, a commercially available microbial product for the treatment of root rot diseases of pea is not currently available.8 
U.S. Pat. No. 5,165,928, issued on Nov. 24, 1992 to Cornell Research Foundation, Inc. and entitled xe2x80x9cBiological Control of Phytophtora by Gliocladiumxe2x80x9d, discloses the use of strains of Gliocladium virens (Syn. Trichoderma virens) on the root biosphere of plants in controlling plant diseases caused by Phyrophthora sojae. More specifically, this patent is directed to the use of specific strains of T. virens on P. sojae-caused stem and root rot in soybean plants.
Steinmetz and Schobeck9 (1993), reported the use of conifer bark inoculum, comprising Trichoderma harzianum or Gliocladium roseum, in controlling Pythium ultimum on pea. Specifically, the applicability of conifer bark as a growth medium and inoculum carrier for T. harzianum or G. roseum was examined, as well as the antagonistic efficacy of such preparations to protect pea seedlings against pre-emergence damping-off caused by P. ultimum. This investigation looked at the possibility of using bark inoculum to control soil. borne pathogens in horticultural practice, and not specifically at the effectiveness of the respective fungal antagonist isolates. However, it was reported that a pretreatment with a nutrient solution was required on the G. roseum-containing bark inoculum for optimum development and efficiency of the antagonist. Accordingly, the G. roseum isolate investigated by Steinmetz and Schobeck is not easily or economically propagated for the treatment of seedlings.
Accordingly, the prior art has not disclosed a biocontrol agent effective against a variety of root rot pathogens, nor is there a bioagent, which can be economically and readily propagated for easy application to seeds or soil. Furthermore, a commercially effective biocontrol agent does not exist which can be applied to a seed or a suitable host, and provide prolonged effectiveness against a range of plant pathogens. In addition, there has not been previously disclosed a biocontrol agent, which, in the absence of an accompanying fungicide, is effective against a range of root rot pathogens including PRRC pathogens Pythium spp., Fusarium solani f.sp. pisi, Fusarium oxysporum f.sp. pisi, Mycosphaerella pinodes, Rhizoctonia solani, Sclerotina sclerotiorum, Alternaria alternata, and Aphanomyces euteiches; common root rot pathogen Bipolaris sorokiniana; and fursarium head blight pathogen Fusarium graminearum. 
The lack of commercial development in the area of biocontrol products, and more specifically with respect to such products effective against root rot pathogens, may be largely in part due to the need for excessive amounts of the biocontrol agent, or, in the case of seed treatments, the short term effectiveness of the product.10 In addition, the commercial availability of biocontrol agents against plant pathogens has been hampered by the lack of effective delivery systems.11 The most successful biologically-based practices reported to date involve integrated management systems which employ a combination of biological, chemical and cultural control measures.11 Accordingly, it is desirable to produce biocontrol agents which are effective against plant pathogens, under a variety of conditions and for a prolonged period of time. In addition, it is desirable to eliminate the need for chemical treatment in controlling root rot diseases in crop plants. More specifically, with respect to the present invention, it is desirable to produce an economically efficient biocontrol agent which can be readily propagated and applied to a plant source or growth medium to provide prolonged effectiveness against a range of PRRC pathogens. In addition, it is desirable to provide a biocontrol agent effective against a range of soil-borne and seed-borne pathogens in a variety of crop species.
A main objective of the present invention is to provide a biological control agent effective against a variety of plant pathogens, and more specifically, to provide a biological control agent effective against PRRC pathogens of pea as well as other root pathogens of other plant species.
It is a further objective to provide an effective and commercially viable method of propagating the biological control agent of the present invention.
Further still, it is an objective of the present invention to provide a biological control agent which is at least as effective against plant pathogens as existing chemical alternatives, under appropriate conditions, and to provide an effective delivery system for said biological control agent.
It is a further objective of the present invention to provide an effective and commercially viable method of propagating the biocontrol agent of the present invention.
Another objective is to provide a delivery system for the effective application of the biocontrol agent of the present invention to a plant source or growth medium.
The present invention is based on the discovery and isolation of a novel strain of Gliocladium roseum Bainier, shown to have antagonistic effects against several plant pathogens, particularly root rot fungal pathogens in groups of Ascomycetes, Deuteromycetes, Oomycetes and Zygomycetes; as well as in a variety of host species in Asteraceae, Brassicaceae, Chenopodiaceae, Cucurbitaceae, Fabaceae, Poaceae and Solanaceae. For example, when applied to seeds of pea, the isolates of the present invention exhibit antagonistic capabilities against fungal pathogens that, in both controlled environments and field conditions, can be at least as effective as current chemical fungicides.
Thus, according to one aspect of the invention, there is provided a biologically pure culture of a strain of a microorganism G. roseum exhibiting antagonistic effects against a plant pathogen. Preferably, the strain of the microorganism is a strain ACM941 having the identifying characteristics of ATCC #74447 (please refer to the deposit information provided later).
According to another aspect of the invention, there is provided a composition a comprising: a culture of a strain of G. roseum (preferably ACM941) exhibiting antagonistic effects against a plant pathogen; and a delivery medium. e.g. a plant seed.
The invention also relates to seeds coated with a strain of the microorganism (preferably ACM941) and to methods of treating plants and plant seeds, etc., with the strain and compositions containing the strain.
According to a still further aspect of the invention, there is provided a method of propagating the strain of G. roseum (preferably ACM941) exhibiting antagonistic effects against fungal plant pathogens, which comprises incubating spores of said strain in a liquid medium for 3 to 5 days at a temperature in the range of 25xc2x0 C. to 30xc2x0 C. under low light intensity and pHxe2x89xa74.5.
As described below, the strain of the present invention has been shown to display antagonistic effects against 13 pathogens including Bipolaris sorokiniana, Fusarium graminearum, Fusarium oxysporum, Fusarium solani, Mycosphaerella pinodes and Sclerotinia sclerotiorum representing the group of Ascomycetes; Ascochyta spp, Rhizoctonia solani and Alternaria alternata representing Deuteromycetes; Pythium aphanidermatum, Pythium ultimum and Aphanomyces euteiches representing Oomycetes; and Rhizopus sp. representing Zygomycetes. These 13 pathogens cause seed decay, root/foot rot, seedling blight and wilt on 17 crop plants including canola, broccoli, brussel sprouts, cabbage and cauliflower (Brassicaceae), field bean, field pea and sweet pea (Fabaceae), sugar beet and table beet (Chenopodiaceae), cucumber (Cucurbitaceae), marigold (Asteraceae), egg plant, pepper and tomato (Solanaceae), and wheat and barley (Poaceae). These are collectively referred to as pathosystems. In particular, these pathosystems are Rhizoctonia solani on pea (field pea and sweet pea), bean (dry bean and garden bean), beet (sugar beet and table beet), broccoli, brussel sprouts, cabbage, canola, cauliflower, cucumber, egg plant, pepper, tomato and marigold; Sclerotinia sclerotiorum, Alternaria alternata, Fusarium oxysporum, Fusarium solani, Pythium aphanidermatum, Pythium ultimum, Aphanomyces euteiches, Ascochyta spp., Rhizopus sp. and Mycosphaerella pinodes on pea; Bipolaris sorokiniana and Fusarium graminearum on wheat and barley. From these results, it is apparent that the strain of G. roseum has a broad effect on most or all fungal pathogens. The host plants appear to have little effect on the efficacy of ACM941 seed treatments in controlling fungal diseases, as shown by the effectiveness of the biocontrol agent against Rhizoctonia foot/root rot caused by R. solani in pea, bean, beet, broccoli, brussel sprouts, cabbage, canola, cauliflower, cucumber, egg plant, pepper, tomato and marigold. It is therefore predictable that the strain of G. roseum (ACM941) will control the same disease caused by the same pathogen in various field and horticultural crops, vegetables, and ornamental plants.
Besides the tested crops, Rhizoctonia root rot caused by Rhizoctonia solani is also a major disease that causes significant yield loss in Canada and worldwide in 589 genera of plants including field and horticultural crops, vegetables and ornamentals. Similarly, Sclerotinia rot or white mold caused by Sclerotinia sclerotiorum is also a very damaging disease of 177 genera of plants in the United States21; Alternaria alternata is a major pathogen of vegetables, field and horticultural crops and causes disease in 110 genera of plants; Fusarium oxysporium is a common wilt pathogen and causes significant damage in 156 genera of plants; Fusarium solani, Pythium spp. and Aphanomyces euteiches are common seed decay and root rot pathogens and cause diseases in 105, 92 and 8 genera of plants, respectively, Bipolaris sorokiniana is a common root rot and damping off pathogen and causes common root rot in 52 genera of plants; Fusarium graminearum is a common root rot and head blight pathogen in 18 genera of plants; and Mycosphaerella pinodes is a major pathogen of field pea and recorded in 6 genera of plants in the United States.
For the reasons given above, it is believed that the G. roseum strain of the present invention controls the diseases caused by the above pathogens in the indicated genus of plants. Thus, on the basis of the morphological characterization and mycoparasitic activity of the novel G. roseum strain herein disclosed, the effectiveness of this bioagent against fungal pathogens in a range of plant species, not just those herein specifically mentioned, is within the scope of the present invention. Specifically, the effectiveness of the G. roseum strain is not limited to the treatment of fungal pathogens in crop species, but also displays promise as a biocontrol agent of fungal pathogens in horticultural plant species, and other plants.
The primary strain of the present invention is G. roseum ACM941. However, it is further believed that other strains of G. roseum, particularly closely related isolates of G. roseum ACM941, display similar antagonist effects against fungal pathogens of plants. Such a additional strains, their compositions and methods of use are thus included within the scope of the present invention.
Thus, according to a first preferred aspect of the invention, there is provided a biologically pure culture of a microorganism G. roseum ACM941, having the identifying characteristics of ATCC #74447.
In accordance with another preferred aspect of the present invention, there is provided a composition comprising a culture of G. roseum ACM941; and a delivery medium.
In accordance with another preferred aspect of the present invention there is provided a coated seed comprising a coating of G. roseum ACM941.
In accordance with yet another preferred aspect of the present invention, there is provided a method for protecting a plant from fungal infection wherein the method comprises the steps of immersing plant seeds in a composition comprising G. roseum ACM941; and planting the seeds in a suitable growth medium.
In accordance with still another preferred aspect of the present invention, there is provided a method of protecting a plant from fungal infection comprising the steps of contacting said plant during a stage of the growth of said plant with a strain of a microorganism Gliocladium roseum exhibiting antagonistic effects against a fungal plant pathogen.
In accordance with still another preferred aspect of the present invention, there is provided a method of controlling fungal plant pathogens, comprising the steps of: providing a delivery medium comprising a culture of a strain of Gliocladium roseum exhibiting antagonistic effects against a fungal plant pathogen; and delivering said delivery medium to plants.
In accordance with a still further preferred aspect of the present invention, there is provided a method of enhancing the growth and productivity of plants comprising: providing a growth medium containing a strain of Gliocladium roseum exhibiting antagonistic effects against a fungal plant pathogen; and planting plant seedlings or seeds in said growth medium,
The primary strain of the present invention has been determined to have a distinct genotype different from known strains of G. roseum, and a sample of the strain was deposited on behalf of Her Majesty the Queen in Right of Canada (as represented by the Minister of Agriculture and Agri-Food Canada) (the intended original assignee of the present application) under the terms of the Budapest Treaty at the American Type Culture Collection of 10801 University Blvd., Manassas, Va. 20110-2209, USA, on Jun. 2, 1998, under accession number ATCC #74447.
The use of the isolates of the present invention for the control of plant pathogens is highly favored over existing chemical alternatives in that it appears not to have adverse effects on non-target organisms, to cause harm to the environment, or to promote pathogen resistance. In the past, biological control agents have been unsuccessful as a result of their inability to propagate in certain soil environments and to promote their parasitic effect for sufficient durations of time. Further, fungal antagonists applied as a seed coating have not routinely colonized the plant rhizosphere.1 
As already noted, the strain of the present invention displayed versatility in its effectiveness against a range of pathogens, as well as its ability to survive and proliferate in the plant rhizosphere, thus providing a target plant with prolonged protection against invasive pathogens.
In addition, the strain of the present invention can be readily propagated in a timely manner, in both solid media and relatively inexpensive liquid media, and applied as seed coatings without difficulty.
Moreover, the effectiveness of the isolates against a range of pathogens lends confidence in their ability to effectively protect a wide range of crops affected by other soil-borne and seed-borne fungal pathogens studied.
Preliminary studies have indicated the potential for the biocontrol ability of the G. roseum ACM941 strain against several major field crop, vegetable and ornamental diseases, including: rhizoctonia root/foot rot of peas, beans, beets, broccoli, brussel sprouts, cabbage, canola, cauliflower, cucumber, egg plant, pepper, and tomato; common root rot of wheat and barley; seed-borne phase of fusarium head blight of wheat and barley; Sclerotinia rot, ascochyta foot rot, fusarium root rot, fusarium wilt, aphanomyces root rot, pythium seed decay and root rot, seed-borne phase of alternaria blight, and seed-borne phase of mycosphaerella blight of field pea.
Studies of the broader applications of G. roseum ACM94 1 against plant pathogens continue to be conducted in western Canada. It is predicted that these studies will confirm that ACM941 is effective against other crops commonly afflicted by soil-borne and seed-borne fungal pathogens.